本文選題：銅綠假單胞菌 + RgsA��； 參考：《福建醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的: 銅綠假單胞菌是臨床上極為常見的條件致病菌，患代謝性疾病、血液病和惡性腫瘤的患者，以及術(shù)后或某些治療后的患者易感染本菌，具有多重耐藥的特點(diǎn)。尋找銅綠假單胞菌通過何種途徑感知外界的變化，以何種方式改變生理學(xué)過程從而適應(yīng)環(huán)境，一直是該菌的研究熱點(diǎn)之一。 細(xì)菌小RNA(small regulatory RNA)是細(xì)菌中普遍存在的一類長度在40～500個(gè)核苷酸的，具有調(diào)控功能的小分子RNA,簡稱為sRNA(small RNA)，sRNA在廣泛細(xì)菌種群的許多關(guān)鍵生命進(jìn)程中發(fā)揮重要的調(diào)控作用，其調(diào)控作用在數(shù)量上和多樣性上甚至超過了蛋白質(zhì)。迄今已在銅綠假單胞菌中發(fā)現(xiàn)29種sRNA，其中RgsA是由Nicolas González在2008年進(jìn)行全基因組搜索發(fā)現(xiàn)的，存在于銅綠假單胞菌和熒光假單胞菌中的sRNA，推測可能與銅綠假單胞菌抗氧化應(yīng)激反應(yīng)有關(guān)，在銅綠假單胞菌生物膜狀態(tài)下高度表達(dá)。銅綠假單胞菌難治性持續(xù)性感染與生物膜形成聯(lián)系密切。在人類感染性疾病中，約有65%與生物膜有關(guān)，例如牙菌斑、囊性纖維化肺病、反復(fù)發(fā)作的慢性中耳炎、慢性骨髓炎、慢性鼻竇炎及慢性傷口感染等。對本研究擬從sRNA RgsA的基本結(jié)構(gòu)、功能、作用機(jī)制到調(diào)控方式進(jìn)行深入研究，以探討該sRNA在銅綠假單胞菌生命活動中的作用。 方法: （1）本研究以銅綠假單胞菌PAO1菌株作為實(shí)驗(yàn)菌株，根據(jù)公用數(shù)據(jù)庫的序列信息和文獻(xiàn)資料設(shè)計(jì)合成引物，采用5′RACE確定轉(zhuǎn)錄起始位點(diǎn)，結(jié)合Northern blot與計(jì)算機(jī)預(yù)測3′末端轉(zhuǎn)錄終止序列，以確定RgsA的基本轉(zhuǎn)錄本結(jié)構(gòu)。 （2）在此基礎(chǔ)上，PCR擴(kuò)增rgsA同源臂與慶大霉素抗性基因插入自殺質(zhì)粒pEX18-Ap，構(gòu)建rgsA基因敲除載體，利用雙親雜交轉(zhuǎn)化銅綠假單胞菌PAO1，構(gòu)建rgsA基因缺陷株SH2-5。 （3） PCR擴(kuò)增rgsA基因編碼片段，插入pJN105質(zhì)粒阿拉伯糖誘導(dǎo)型啟動子的下游，構(gòu)建rgsA基因表達(dá)載體，轉(zhuǎn)化rgsA基因缺失缺陷株SH2-5，構(gòu)建rgsA基因補(bǔ)償/過表達(dá)株。 （3）以生長時(shí)間為橫坐標(biāo)，OD600值為縱坐標(biāo)繪制生長曲線，觀察野生株、rgsA基因缺陷株的生長情況。（4）利用Trizol法分別抽提不同濃度過氧化氫，有機(jī)過氧化物刺激下銅綠假單胞菌的總RNA，Real-time PCR檢測RgsA表達(dá)情況。 （5）以含有不同濃度過氧化氫，有機(jī)過氧化物L(fēng)B平板作為培養(yǎng)基進(jìn)行平板實(shí)驗(yàn)，以不同濃度過氧化氫與不同反應(yīng)時(shí)間組合，進(jìn)行MTT實(shí)驗(yàn)，比較野生株，缺失株以及過表達(dá)菌株在浮游菌狀態(tài)下抗氧化應(yīng)激能力。 （6）野生株，缺失株以及過表達(dá)菌株分別進(jìn)行生物膜培養(yǎng)，三天后利用不同濃度過氧化氫分別對形成的生物膜進(jìn)行刺激， SYTO9/PI染色后熒光顯微鏡觀察，以比較三者間在生物膜狀態(tài)下抗氧化應(yīng)激能力。 結(jié)果： （1）rgsA在銅綠假單胞菌中存在兩個(gè)轉(zhuǎn)錄本，轉(zhuǎn)錄本長度分別約為300bp與90bp。其中短片段轉(zhuǎn)錄本的轉(zhuǎn)錄起始位點(diǎn)，位于NCBI注釋基因片段起始下游86bp處，長片段轉(zhuǎn)錄本的轉(zhuǎn)錄起始位點(diǎn)，位于NCBI注釋基因片段起始上游157bp處。 （2）經(jīng)PCR及測序證明，銅綠假單胞菌缺陷株SH2-5中缺失rgsA基因，成功構(gòu)建銅綠假單胞菌rgsA基因缺陷株。經(jīng)測序證明，過表達(dá)質(zhì)粒pJN105-SH和補(bǔ)償/過表達(dá)菌株SH2S構(gòu)建的正確性。 （3）生長曲線提示rgsA基因缺陷株生長速度明顯低于野生株。 （4）經(jīng)Real-time PCR檢測，不同濃度過氧化氫以及有機(jī)過氧化物刺激下，RgsA表達(dá)均有不同程度的增加，尤其在有機(jī)過氧化物刺激時(shí)表達(dá)明顯上升。 （5）通過計(jì)算生長于含有不同濃度過氧化氫與有機(jī)過氧化物的LB平板上的菌落數(shù)，以及MTT法檢測各實(shí)驗(yàn)組中細(xì)菌總體活性，rgsA基因缺陷株表現(xiàn)出對過氧化氫及有機(jī)過氧化物的抵抗能力明顯下降，而在突變菌株中導(dǎo)入表達(dá)載體后，在0.1%L-Arabinose的誘導(dǎo)下，對氧化應(yīng)激的抵抗能力恢復(fù)到接近野生株的水平。 （6）野生株，缺失株，，過表達(dá)株在流動培養(yǎng)系統(tǒng)中培養(yǎng)3天后形成生物膜，在30mM過氧化氫刺激下，相比野生株及0.1%L-Arabinose的誘導(dǎo)下的過表達(dá)株細(xì)菌，缺失株出現(xiàn)大面積能被SYTO9染色的死菌（呈黃色）。 結(jié)論： rgsA在銅綠假單胞菌中存在兩個(gè)轉(zhuǎn)錄本，證明RgsA在銅綠假單胞菌抗氧化應(yīng)激中發(fā)揮重要作用。
[Abstract]:Objective:
Pseudomonas aeruginosa is a very common pathogenic bacteria in clinic. Patients with metabolic diseases, hematological and malignant tumors, and patients after or after some treatment are susceptible to the bacteria and have the characteristics of multiple drug resistance. To adapt to the environment, it has always been one of the research hotspots of the bacteria.
Bacterial small RNA (small regulatory RNA) is a common type of nucleotides, a small molecule with 40~500 nucleotides and regulatory functions, RNA, called sRNA (small RNA). SRNA plays an important regulatory role in many critical life processes in a wide range of bacterial populations, and its regulatory role is even in quantity and diversity. To date, 29 kinds of sRNA have been found in Pseudomonas aeruginosa, of which RgsA was found in the whole genome search by Nicolas Gonz ah lez in 2008, existed in Pseudomonas aeruginosa and sRNA in Pseudomonas fluorescens, presumably related to the antioxidant stress response of Pseudomonas aeruginosa and high in the state of Pseudomonas aeruginosa biofilm. In human infectious diseases, about 65% are associated with biofilms, such as plaque, cystic fibrosis, chronic otitis media, chronic osteomyelitis, chronic sinusitis, and chronic wound infection. This study is intended to be based on the base of sRNA RgsA. This structure, function, action mechanism and regulation mode were studied in depth to explore the role of sRNA in the life activities of P. aeruginosa.
Method:
(1) in this study, Pseudomonas aeruginosa PAO1 strain was used as an experimental strain. The primers were designed according to the sequence information and literature of the public database. The transcriptional starting site was determined by 5 'RACE, and the 3' terminal transcriptional terminating sequence was predicted with Northern blot and computer to determine the basic transcriptional structure of RgsA.
(2) on this basis, the rgsA homologous and gentamicin resistant genes were inserted into the suicide plasmid pEX18-Ap, and the rgsA gene knockout vector was constructed by PCR, and the rgsA gene defective strain SH2-5. was constructed by using parental hybridization to transform the PAO1 of Pseudomonas aeruginosa.
(3) PCR amplified rgsA gene encoding fragment, inserted the downstream of the pJN105 plasmid Arabia sugar inducible promoter, constructed the rgsA gene expression vector, transformed the rgsA gene deletion defect strain SH2-5, and constructed the rgsA gene compensation / overexpression strain.
(3) taking the growth time as the horizontal coordinate and the OD600 value as the longitudinal coordinate to draw the growth curve to observe the growth of the wild plant and the rgsA gene defective strain. (4) the Trizol method was used to extract the different concentration of hydrogen peroxide, and the total RNA of Pseudomonas aeruginosa was stimulated by organic peroxide, and the Real-time PCR was used to detect the expression of RgsA.
(5) the experiment was carried out with different concentrations of hydrogen peroxide and organic peroxide LB plate as medium. The MTT experiment was carried out at different concentrations of hydrogen peroxide and different reaction time. The anti oxidative stress ability of wild plant, missing strain and overexpressed strain in the state of planktonic bacteria was compared.
(6) wild plants, missing strains and overexpressed strains were cultured for biofilm respectively. After three days, the biofilms were stimulated by different concentrations of hydrogen peroxide. After SYTO9/PI staining, the fluorescence microscope was observed to compare the antioxidant activity of the three in the biofilm state.
Result錛

本文編號：1973437